Process for the maintenance and proliferation of defective non-infectious virus genomes in proliferating plant materials

ABSTRACT

A process for the maintenance and proliferation of defective, non-infectious viruses or virus genomes, comprising mutations, i.e. deletions, substitutions, insertions and new rearrangements of the viral genes or combinations of the virus DNA with heterologous genetic material in proliferating plant material and for the regeneration of whole plants containing stably integrated into their genome said defective, non-infectious virus DNA.

This application is a continuation-in-part of application Ser. No.465,512, filed Jan. 16, 1990, now abandoned, which is acontinuation-in-part of application Ser. No. 07/074,593, filed Jul. 17,1987, now abandoned, which is a continuation-in-part of application Ser.No. 06/636,946, filed Aug. 2, 1984, now abandoned.

FIELD OF THE INVENTION

The present invention relates to a process for the maintenance andproliferation of defective, non-infectious viruses or virus genomes,comprising mutations, i.e. deletions, substitutions, insertions and newrearrangements of the viral genes or combinations of the virus DNA withheterologous genetic material, in proliferating plant material and to aprocess for the regeneration of whole plants containing stablyintegrated into their genome said defective, non-infectious virus DNA.

Plant material with novel or improved properties can be produced byusing genetically manipulated viruses as vectors when inserting newgenetic information into plant hereditary material.

Owing to the rapid increase in world population, the geneticmodification of useful plants is a major point of focus of biologicalresearch. On the one hand there is the search for alternativereproducible sources of food, energy and raw materials, e.g. new plantspecies, especially hybrid species with valuable properties such asincreased resistance to pathogens (e.g. phytopathogenic insects, fungi,bacteria, viruses etc.), to herbicides, insecticides or other biocides,to atmospheric influences or location conditions (e.g. heat, cold, wind,soil condition, moisture, dryness etc.), or with increased formation ofreserve or storage substances in leaves, seeds, tubers, roots, stalksetc. On the other hand, in addition to the growing need for valuablebiomass, there is also an increased need for pharmaceutically acceptableactive ingredients of plant origin and derivatives thereof, e.g.alkaloids, steroids and the like, which, because of the low yield fromnatural sources, are being increasingly made available by alternativemethods, for example by extraction from genetically manipulated plantspecies.

Accordingly, there is growing interest in the practical possibility ofselectively manipulating numerous plant species.

One means of attaining this object is the transfer of foreign genes toisolated plant cells, the replication and expression of the geneticmaterial, as well as its propagation and maintenance in all daughtercells formed by cell division from the initial cell. Such daughter cellsmay exist in the form of single cells, tissue cultures or whole plants.

Dividing plant cells have a tendency to block the infiltration ofviruses and to inhibit, partially or completely, the viability andmultiplication capacity of viruses.

For example, it is known that cauliflower mosaic virus (CaMV) is able toproliferate in the differentiated cells of a plant and to infect allother differentiated cells, but the growth centres or meristems, i.e.the dividing cells, are not infected. Thus virus-free plants can beregenerated from virus-infected plants through a meristem culture.

Additionally it has been supposed that for the transformation ofhereditary plant material using viruses as vectors, and for theproduction of genetically identical progeny of the initial plantmaterial, it is necessary to cultivate viruses without loss of theirability to self-replicate and infiltrate new host cells.

It was therefore to be expected that the use of viruses as vectors forthe transfer of new genetic information in many cases would not lead togenetically transformed plant material, as the viruses either do notinfiltrate proliferating plant material or would die therein, or geneticmaterial which is inserted into proliferating plant cells through plantviruses would not be absorbed into the hereditary material of the plantcells or would not replicate. According to Kriedel and Goodman [J. C.Kriedel and R. M. Goodman, BioEssays Vol. 4(1), pp. 4-8, (1986)], forexample, ". . . caulimoviruses do not integrate into the chromosomal DNAof their host".

Within the scope of the present invention this prejudice could beovercome.

Prior to the present invention, in constructing a vector-system for thetransformation of plants it was necessary to make a choice between aviral vector and an integration-type vector such as the ti-plasmidspecifically, the T-DNA border regions of the Ti-plasmid.

A viral vector could only be used for a systemic infection of the hostplant whereupon an integration type vector delivers its foreign geneticmaterial into the plant chromosome and, therefore, is also only usefulin dividing plant material. [R. E. Gardner, Plant Viral Vectors: CaMV asan Experimental Tool, (pp. 123-124, viral versus integration vectors)in: Genetic Engineering of Plants, Kosue et al., ed. Plenum Press p.p.121-127 (1982)]. Now by use of the present invention the possibilityopens up to utilize plant viruses, particularly plant viruses withoutthe T-DNA border regions of the Ti-plasmid as integration-type vectorstoo.

Surprisingly, it has been found that it is possible to cultivatedefective, non-infectious viruses or defective, non-infections virusgenomes without the T-DNA border regions of the Ti-plasmid comprisingmutations, i.e. deletions, substitutions, insertions and newrearrangements of the viral genes and combinations of the virus DNA withheterologous genetic material in dividing, i.e. proliferating, plantmaterial and to insert said virus genomes into plant genomes.

By carrying out the process of the present invention it is now possibleto cultivate defective, non-infectious virus genomes in dividing, i.e.proliferating plant material (protoplasts, cells or tissues) and stablymaintain and proliferate the mutant viral genome therein (see example5). It has been found that the viral DNA is integrated into and stablymaintained in the plant genome. The present invention therefore opens upa lot of new possibilities to manipulate plant material.

So far it was only possible to integrate very small genes or genefragments (up to 500 base pairs) into the viral genome, for example ofCaMV, because there are only a few sites around the viral genome, whichare not essential for infectivity and which are therefore suitable forthe exchange with foreign DNA material. Thus, by carrying out theprocess of the present invention, it is possible to integrate normallysized genes (up to 1200 base pairs, like the kanamycin-resistance gene)into the deletion site of the defective viral genome, transform plantprotoplasts, cells or tissues and stably maintain and proliferate theforeign genetic material together with the mutant viral genome inproliferating plant material.

There are substantially no limitations with respect to the extent of theinserted genetic information and plants, which contain such defectiveviruses or their genomes exhibit no symptoms of disease.

SUMMARY OF THE INVENTION

Accordingly, the present invention relates to a process for themaintenance and proliferation of defective, non-infectious virus genomescomprising mutations, i.e. deletions, substitutions, insertions and newrearrangements of the viral genes and combinations of the virus DNA withheterologous genetic material in proliferating plant material, whichprocess comprises cultivating isolated plant protoplasts, cells ortissues which contain said virus genomes without the T-DNA borderregions of the Ti-plasmid in a suitable culture medium. The process ofthis invention also encompasses the cultivation and regeneration ofwhole plants. Some of the plants so obtained are resistant to furthervirus infections (cross-resistance).

It has been known for a long time, that inoculation of plants with mildstrains of viruses or viroids prevents more virulent strains of the sameor related virus strains from infecting said plants and causing moresevere disease symptoms.

This phenomenon, referred to as "cross-protection" and first discoveredby McKinney (1929) with tobacco mosaic virus (TMV), has been used toprotect certain crop plants such as tomatoes, potatoes and citrusagainst attack of tomato mosaic virus, potato spindle tuber viroid andcitrus tristeza virus, respectively in order to reduce yield losses[Brodbent, L., 1976; Fernow, K. H., 1967; Costa, A. S. and Muller, G.W., 1980].

Nevertheless infection of healthy crop plants with mild strains ofpathogenic viruses to confer "cross-resistance" to said plants, containsmany risks, particularly the possibility of causing an epidemic disease,provided, for example, the used mild virus strain has mutated into avirulent form.

Within the scope of the present invention it is now possible to confer"cross-resistance" to plants without previously infecting them usingwild virus variants of pathogenic strains, avoiding the risk of causinga disease outbreak.

In one embodiment of this invention plants are regenerated from plantprotoplasts, cells or tissues containing integrated into their genomedefective non-infectious viral DNA or DNA fragments, said viral DNA orDNA fragments conferring "cross-resistance" to the regenerated plant.

Within the scope of this invention it is therefore possible to usenon-infectious viruses which are not viable under natural conditions,particularly non-infectious viruses without the T-DNA border regions ofthe Ti-plasmid, (defective viruses) and have substantially no limitationwith respec to the extent of the inserted genetic information, or thegenomes thereof, such that the plants which contain such viruses ortheir genomes exhibit no symptoms of disease. Hence defective virusescan be used with particular advantage as vectors for geneticinformation. Further, it is possible to regenerate whole plants, whichare resistant to pathogenic viruses, from protoplasts, cells or tissueswhich contain defective viruses or their genomes.

Within the scope of this invention, some terms commonly used inrecombinant DNA and plant genetics technology are utilized.

In order to provide a clear and consistent understanding of thespecification and claims, including the scope given such terms, thefollowing definitions are provided:

Virus Genomes: genetic information of a virus in the form of

original DNA

original RNA

a transcript

cDNA

Defective, non-infectious Viruses or defective non-infectious VirusGenomes:

viruses or virus genomes which are not viable under natural conditionsand which have lost their ability to replicate in plant material and,therefore, to infect their host plants systemically on account ofdeletions, mutations, new combinations or combinations with new geneticmaterial. The foregoing is inclusive of viruses or viruses genomeswithout the T-DNA border regions of the Ti-plasmid.

Plant Promoter: A DNA expression control sequence that is capable ofcausing the transcription in a plant of any homologous or heterologousDNA genetic sequence operably linked to such promoter.

Plant: Any photosynthetic member of the kingdom Planta that ischaracterized by a membrane-bound nucleus, genetic material organizedinto chromosomes, membrane-bound cytoplasmic organelles, and the abilityto undergo meiosis.

Plant Cell: The structural and physiological unit of plants, consistingof a protoplast and cell wall.

Plant Tissue: A group of plant cells organized into a structural andfunctional unit.

Plant Organ: A distinct and visibly differentiated part of a plant suchas root, stem, leaf or embryo.

Protoplast: isolated plant cell without cell walls, having the potencyfor regeneration into a cell culture or a whole plant

Cell Culture: proliferating mass of cells in a undifferentiated state.

DESCRIPTION OF THE FIGURE

FIG. 1: Constructs used for transformation

Restriction sites:

    B=BstEII, E=EcoRV, H=HindIII, S=SalI, Sp=SphI.

1051: Control construct containing the Km gene.

1050: 1.1 mer genome of wild type CaMV-DNA [a hybrid of strains 4-184(genes VII-V) and S (Gene VI)]. This construct allows for transcriptionof genomic RNA. Inoculated turnips show vein clearing symptoms on theleaves (FIG. 6A).

1123: 1.1 mer genome of wild type CaMV-DNA [a hybrid of strains DH(genes VII-V) and S (gene VI)], which needs to recombine in order tobuild viable virus. This construct induced strong stunting of inoculatedturnips and the leaves of infected plants turned pale green with mosaicor mottle type symptoms (FIG. 6A).

1108: 1.3 mer genome of CaMV (strain S) with an in frame deletion withingene III.

1105: 1.3 mer genome of CaMV (strain S) with an out of frame deletionwithin gene III.

1016: CaMV gene VI construct. CaMV strain S DNA. Agroinfection withstrains 1108, 1105 and 1016 did not result in symptom formation onturnip plants.

LB/RB: Left, respectively, right border of the Ti-plasmid.

Nop: Nopaline synthase gene; located in the T-DNA of A. tumefaciens(nopaline strains).

pBR322: Complete sequences cut at HindIII site. Replace the oncogenicfunctions in Ti plasmid pGV3850 (Zambryski et al., 1983).

pHC79: Sequences from BstEII to HindIII.

cos: Cosmid sequences from phage lambda. Tn903: Kanamycinphosphotransferase gene of transposon Tn 903.

Km: aminoglycoside phosphotransferase II gene from transposon Tn5. TheSalI-BstEII fragment, containing the Km gene is derived from pCaMV6km(Paszkowski et al., 1986).

I-VII: CaMV genes (boxes with dotted pattern).

19S/35S: Transcripts starting at the 19S or at the 35S promoter.

DETAILED DESCRIPTION OF THE INVENTION

Suitable initial plant materials for the process of the invention are inparticular isolated protoplasts, preferably those of agriculturallycultivated plants. Suitable cultivated plants in this context are inparticular those of the families Gramineae, Solanaceae, Compositae andCruciferae and of the order Leguminosae.

Representatives of the aforementioned group are e.g. for Gramineae:wheat, rye, barley, oats, rice, maize, sorghum and sugar cane; forSolanaceae: potatoes, tomatoes and tobacco; for compositae: sun flowers,lettuce (Lactuca sativa), endive (Cichorium endivia) and camomile(Matricaria); for Cruciferae: various cabbage and beet varieties and oiland spice plants; and for Leguminosae: soybeans, lupins, lucernes, peas,beans and groundnuts.

Among the Cruciferae the genus Brassica, comprising e.g. rape, turnip,black mustard, white mustard, and cabbage and beet varieties, is to besingled out for special mention, in particular Brassica rapa, forexample Brassica rapa cv Just Right and Brassica napus. A preferredembodiment of the process of the invention comprises culturing isolatedplant protoplasts, cells or tissues which contain genomes of defective,non infective plant viruses.

According to another preferred embodiment of the process of thisinvention, the isolated plant protoplasts, cells or tissues containdefective, non-infectious virus genomes, which contain genetic materialthat is inserted into the plant genome of said protoplasts, cells ortissues. The viruses are in particular DNA viruses, preferablycaulimoviruses, with cauliflower mosaic virus being most preferred.

Virus genomes which merit attention are primary viral DNA and DNA copiesof viral RNA. The virus genomes can be genetically manipulated. Forexample, they can be restricted in their infectivity and theirpathogenicity or contain inserted, foreign genetic material.

Genes which may be used in this invention may be homologous orheterologous to the plant cell or plant being transformed. It isnecessary, however, that the genetic sequence coding for (a) desiredproteinaceous product(s) be expressed, and produces a functional enzymeor polypeptide in the resulting plant cell. Thus, the inventioncomprises plants containing either homologous genes or heterologousgenes that express desired proteinaceous product(s). Further, theheterologous gene(s) may be from other plant species, or from organismsof different kingdoms, such as microbs or mammals or they may be ofsynthetic origin.

In general it is expedient to culture isolated plant protoplasts, cellsand tissues which contain virus genomes with inserted foreign geneticmaterial of plant origin, preferably material of a plant whose genomediffers from that of the genome of those protoplasts, cells or tissueswhich contain the virus genomes, i.e. that genetically different plantsact as recipients and donors of the genetic material.

Primarily, gene(s) or genetic material are contemplated in thisinvention, which provide the transformed plant protoplasts, cells ortissues with valuable properties, such as increased resistance topathogens (e.g. phytopathogenic insects, fungi, bacteria, viruses etc.),to herbicides, insecticides or other biocides, to atmospheric influencesor location conditions (e.g. heat, cold, wind, soil condition, moisture,dryness etc.), or with increased formation of reserve or storagesubstances in leaves, seeds, tubers, roots, stalks etc.

Also included in the present invention are genes coding forpharmaceutically acceptable active ingredients, e.g. alkaloids,steroids, hormones and other physiologically active substances.

Therefore genes, which are contemplated in this invention include, butare not limited to, plant specific genes, such as the zeine gene[Wienand, U., et al., Mol. Gen. Genet, 182: 440-444 (1981)] mammalianspecific genes, such as the insulin gene, the somatostatine gene, theinterleucine gens the t-PA-genes [P-Pennica. et al., Nature 301, 214(1983)] etc., or genes of microbial origin, such as the NPT II gene aswell as gene of synthetic origin such as the insulin gene [Stephien, P.,et al., Gene, 24: 281-297 (1983)] and the somatostatin gene [K. Itakuraet al., J. Am. Chem. Soc, 97: 7327 (1975)].

At present some 300 plant viruses are known, which are divided into 25different groups.

Viruses which can be used in this invention as vectors for theintroduction of chimaeric genes into plant material are those which aresuitable on account of their biological properties i.e. their hostrange, their virulence, their ease of mechanical transmission, theirrate of seed transmission etc. and whose genetic material can bemanipulated and reintroduced into the plant in a biologically activeform, i.e., the DNA clones of said viruses must be infectious.

Viruses which are contemplated in this invention include, but are notlimited to, members of the cauliflower group, including cauliflowermosaic virus (CaMV), and members of the geminiviruses whose genome isssDNA including bean golden mosaic virus (BGMV), chloris striate mosaicvirus (CSMV), cassava latent virus (CLV), curly top virus (CTV), maizestreak virus (MSV) and wheat dwarf virus (WDV).

Also included in the present invention are RNA-containing plant viruses,which are able to be manipulated genetically using cDNA copies of theirRNA genome.

Suitable carriers for the inserted foreign genetic material are inparticular DNA viruses, preferably caulimoviruses, and of these mostpreferably cauliflower mosaic virus.

A particularly preferred embodiment of the process of the inventioncomprises culturing isolated plant protoplasts, cells or tissues ofBrassica rapa, for example Brassica rapa cv Just Right, which containgenomes of cauliflower mosaic virus with inserted foreign geneticmaterial of a plant whose genome is different from the genome of theprotoplasts, cells or tissues of Brassica rapa.

The introduction of defective, non-infectious virus DNA, morespecifically, defective, non-infectious virus DNA without the T-DNAborder regions of the Ti-plasmid, into plant protoplasts and theculturing of the isolated plant protoplasts, cells or tissues containingsaid virus DNA can be carried out by known methods or by methodsanalogous to known ones.

Isolated plant protoplasts which constitute suitable starting materialfor isolated cells and tissues can be obtained from any parts of theplant, for example from leaves, stems, blossoms, roots, pollen,cotyledons, seedlings or seeds. It is preferred to use leaf protoplasts.The isolated protoplasts can also be obtained from cell cultures.Methods of isolating protoplasts may be found for example in Gamborg, O.L. and Wetter, L. R., Plant Tissue Culture Methods, 1975, 11-21.

The working up and culturing of the plant material is convenientlyperformed in accordance with the following particulars, but withoutimplying any restriction thereto.

The plant organs--provided the starting material does not consist ofalready sterile cell cultures--must be sterilised, for example bytreatment with ethanol, HgCl₂, Ca(OCl)₂, or a commercial bleachingagent, and then washed carefully in sterile water. All subsequentmanipulations must be carried out under absolutely sterile conditions.Sterility is also a requirement for the equipment and solutionsemployed.

The degradation of the cell walls of tissue aggregates is effectedenzymatically, for example by using cellulases, hemicellulases orpectinases which are commercially available as microbial culturefiltrates and are used in the concentration range of 0.001 to 5%(throughout this specification percentages are by weight, unlessotherwise indicated).

During isolation and subsequent culturing, the turgor pressure of theplant cells must be equalised by a higher osmotic pressure of theisolation medium and of the culture medium. This is done by usingosmotically active substances such as mannitol, sorbitol, saccharose,glucose, CaCl₂ or other neutral salt ions, alone or in combination withone another and/or with simple culture media. The osmotic pressure ofthe isolation and culture media is conveniently in the range from about200 to 1000 mOs/kg H₂ O (Os=molality=mole of osmotically activeparticles/kg of solvent).

The enzyme mixtures are normally adjusted to pH values in the range from5.2 to 8.0, preferably to pH 5.4.

Incubation of the tissues, which are conveniently cut into fine sections(when using leaves, the epidermis can also be removed), is effected ingeneral in the temperature range from 5° to 40° C., normally at 24° C.The incubation time depends on the concentration of the enzymes, theincubation temperature, the type of tissue and the state ofdifferentiation of the tissue. The incubation time is normally from 20minutes to several days.

After incubation, the protoplasts are isolated by passing the incubatedmaterial through a sieve with a mesh size between 25 and 280 μm toremove undigested tissue, and concentrated by centrifuging. Theprotoplasts sediment or float, subject to their own buoyant density andto the density of the incubation and wash solution. The sedimenting orfloating protoplasts are washed repeatedly by resuspending andcentrifuging them in osmotically stabilised wash media (e.g. solutionsof sugar alcohols, sugars or neutral salts, alone or combination withone another and/or culture media; pH in the range from 5.4 to 6.5) andsubsequently taken up in culture media. An important factor in thesubsequent culturing is the population density of the protoplasts, i.e.the number of protoplasts per milliliter of culture medium. Thepopulation density is advantageously in the range from 1 to 1×10⁶ /ml,with the optimum range being from about 2×10⁴ /ml to 6×10⁴ /ml.

The methods of culturing can vary from culturing in a liquid culturemedium as thin layer in petri dishes, through drops or suspending dropswith volumes in the range from about 1 μl to 200 μl, to culturing insolidified culture media (agar, agarose), or in combinations ofsolidified and liquid culture media. Culturing can be carried out inpetri dishes, multiliter plates, containers, Erlenmeyer flasks,microcapillaries or tissue culture flasks which may be of coated oruncoated plastic or of glass.

Culturing can be carried out normally in a temperature range from 7° to42° C., preferably in the range from 24° to 27° C. The temperature caneither be kept constant or varied sequentially.

The light conditions during culturing may vary from permanent darknessto permanent light in the range from 500 to 5000 lux and alternatingperiods of light/darkness. Alternating sequences of light/darkness arepreferred.

There is already available a wide range of culture media, which differin individual components or groups of such components. However, thecomposition of all media is in accordance with the following principle:they contain a group of inorganic ions in the concentration range fromabout 10 mg/l to several hundred mg/l (so-called macroelements, e.g.nitrate, phosphate, sulfate, potassium, magnesium, iron), a furthergroup of inorganic ions in maximum amounts of several mg/l (so-calledmicroelements, e.g. cobalt, zinc, copper, manganese), and a number ofvitamins (e.g. inositol, folic acid, thiamine), a source of carbon andenergy such as sucrose or glucose, and growth regulators in the form ofnatural or synthetic phytohormones from the classes of auxins andcytokinins in the concentration range from 0.01 to 10 mg/l. The culturemedia are additionally osmotically stabilised by the addition of sugaralcohols (e.g. mannitol) or sugar (e.g. glucose) or salt ions (e.g.CaCl₂), and are adjusted to a pH value of 5.6 to 6.5.

A more detailed description of commonly employed culture media can befound e.g. in Koblitz, H., Methodische Aspekte der Zell- undGewebezuchtung bei Gramineen unter besonderer Berucksichtigung derGetreide, Kulturpflanze XXII, 1974, 93-157.

The culturing and incubation conditions such as temperature of theculture medium and ambient temperature, humidity, light intensity andduration thereof, replenishment or regeneration of culture media orchange in their composition, can be adapted to the respective conditionssuch as nature of the protoplasts, type of virus genomes, rate of growthand division of the plant material and/or of the virus genomes, amountof debris and other factors.

Culturing of the isolated plant protoplasts, cells or tissues is carriedout in a suitable culture medium, normally in a solidified culturemedium.

Agarose can be used with pre-eminent success as gelling agent. Thecontent of agarose in the nutrient medium is normally from 0.4 to 4%.

Agarose is one of the constituents of agar. Commercially available agarconsists mainly of a mixture of neutral agarose and ionic agaropectinwith a large number of attached side groups. Commercial agarose isobtained from agar by conventional commercial methods. Usually a numberof side chains remain intact and determine the physicochemicalproperties such as gel formation and melting and gelling temperature.

Agarose which melts and gels at low temperature is obtained e.g. bysubsequently introducing hydroxyethyl groups into the agarose molecule.Agarose modified in this manner shall be referred to throughout thisspecification as LMT (low-melting) agarose.

Examples of suitable agarose preparations are the following types: SeaPlaque LMT®, LMP®, type VII®, HGT®, HGTP®, LE Standard LMT® orSea-Prep®.

The protoplasts, cells or tissues containing the virus genomes or theirderivatives can be inserted into the culture medium in various ways. Forexample, the procedure can be that protoplasts, cells or tissuescontaining the defective, non-infectious viruses or defective,non-infectious virus genomes are put into a liquid, warm culture mediumwhich already contains the gelling agent and which solidifies uponcooling. However, it is more advantageous to add a further portion ofthe liquid culture medium with the gelling agent to a suspension of theprotoplasts, cells or tissues containing the virus genomes or theirderivatives in a liquid culture medium without gelling agent and, afterthorough mixing, to allow the medium to solidify.

Particularly good results are obtained by surrounding the solidifiedculture medium containing the protoplasts, cells or tissues whichcontain the defective, non-infectious viruses or defective,non-infectious virus genomes with liquid culture medium. Thus, forexample, the solidified culture medium, after it has been plated in thedishes and plates, e.g. petri dishes, which are also used for culturingbacteria or yeast cells, can be cut into "segments" after it hassolidified and the segments transferred to a liquid culture medium,preferably a nutrient solution. The term "segment" in the context ofthis invention denotes a three-dimensional irregular or, preferably,regular structure, e.g. discs, spheres, cubes, prisms, cones and thelike, having an average cross-section of 1 to about 100 mm, preferably 2to 60 mm, and most preferably, 2 to 10 mm. The volume ratio of segmentsto liquid culture medium or nutrient solution is advantageously in therange from 1:20 to 1:100. It is advisable to agitate the liquid culturemedium containing the segments, for example by shaking on a gyratoryshaker. The segmentation of the solidified medium is usually effectedimmediately after the medium has set or within a period of 4 weeks,preferably of 0 to 8 days after the medium has set, depending on theprotoplast type. The best time for petunia protoplasts is the 4th day,for tobacco and crepis capillaris the 3rd day, after plating. Whenplating Brassica rapa protoplasts, segmentation is most preferablyeffected immediately after gelation of the agarose.

Segments are prepared which are preferably homogeneous in size and form.They may have an irregular or, preferably, regular structure, e.g.cones, cubes, prisms, discs and spheres. The segments have in general anaverage diameter or cross-section of 2 to 100 mm, preferably from 2 to10 mm. For further details reference is made to EP-A 129,668.

Within the scope of the process according to the invention, it ispossible to use both protoplasts, cells and tissues already containingdefective, non-infectious viruses or defective, non-infectious virusgenomes before being isolated, or those which were still virus-freebefore their isolation and are later artificially provided with thedefective, non-infectious viruses or defective, non-infectious virusgenomes.

A variety of methods for the introduction of non-infectious DNA-virusesinto plant material are known in the art. One of these methods comprisesthe use of lipid vesicles, so-called liposomes, to encapsulate thegenetic material followed by the fusion of said liposomes with the plantprotoplast and the release of the encapsulated genetic material into theprotoplast cytoplasm. (Lurquin PF, 1979). A further approach comprisesthe microinjection of genetic material contained in the vector directlyinto the plant cells by use of micro-pipettes to mechanically transferthe recombinant DNA.

In an alternate embodiment of this invention the DNA may be transferredinto plant protoplasts after previously having been complexed witheither polycationic substances such as, for example, poly-L-ornithine(Davey et al., 1980) or calcium phosphate (Krens et al., 1982) and/orafter treatment of the protoplasts with polyethylene glycol (Paszkowskiet al., 1984).

In particular this method involves contacting the DNA probe, comprisingthe probe molecule as well as a carrier DNA, with plant protoplast in asuitable osmotically stabilized incubation medium, such as, for example,but not limited to, K₃ -medium (Nagy JI and Maliga R, Z.Pflanzenphysiologie 78: 453-455, 1976; Shillito RD et al., MutationResearch 81: 165-175, 1981), containing polyethylene glycol with amolecular weight preferably between 1000 and 10000 g/Mol, morepreferably between 3000 and 8000 g/Mol, and in a concentration rangefrom 5% to 30% (w/v). The protoplasts and the DNA probe may be incubatedfor a period of 10 to 60 min, preferably for a period of about 30 min.

The incubation may be carried out in the light or, preferably, in thedark at a temperature between 18° C. and 32° C., preferably between 22°C. and 28° C.

The process of the present invention also encompasses the maintenanceand proliferation of defective, non-infectious viruses or defective,non-infectious virus genomes, comprising mutations, i.e. deletions,substitutions or new rearrangements of the viral genes and combinationsof the virus DNA with heterologous genetic material by culturing theisolated plant protoplasts, cells or tissues containing said viruses orvirus genomes in a suitable culture medium. The virus mutants may bethose which are not viable under natural conditions, in particularmutants of plant viruses.

Mutants, which are not viable under natural conditions and which arecontemplated in this invention include, but are not limited to, those,which are produced by insertion deletion or substitution mutagenesis,resulting in destruction of distinct regions of the viral genome, whichare essential for infectivity, pathogenicity, and viability of thevirus.

Insertions of a 12 bp fragment, for example, which cause an insertion of4 amino acids in the resulting protein product made in open readingframe (ORF) I, III, IV (coat protein gene) and V of the CaMV genomeproved to be lethal [Gardner et al., 1982].

The protoplasts, cells or tissues can contain for example primary viralDNA of such virus mutants which are not viable under natural conditions,or can contain corresponding mutants of such DNA viruses, representativetypes of DNA viruses being in particular caulimoviruses, especiallycauliflower mosaic virus.

Also falling within the scope of this invention are regenerated plantcells, plant tissues or whole plants which contain defective,non-infectious viruses or defective, non-infectious virus genomes,provided they have been produced from isolated plant protoplastscontaining these virus genomes in particular from protoplasts ofcultivated plants.

Suitable cultivated plants in this context are in particular those ofthe families Gramineae, Solanaceae, Compositae and Cruciferae and of theorder Leguminosae. Plants of the family Cruciferae to be singled out forspecial mention are those of the genus Brassica, in the present contextpreferably Brassica rapa, for example Brassica rapa cv Just Right.

Viruses are in particular plant viruses.

The regenerated plant cells, plant tissues or whole plants can containfor example primary defective, non-infectious viral DNA or defective,non-infectious DNA viruses, especially caulimoviruses and, mostparticularly, cauliflower mosaic virus. Regenerated plant cells ortissues of Brassica rapa which contain cauliflower mosaic virus are tobe particularly mentioned.

In addition, the regenerated plant cells or tissues can contain thosedefective, non-infectious virus genomes thereof which carry geneticmaterial which is inserted into the genome of the plant cells ortissues; or they can contain genetically manipulated virus genomes, forexample those which contain inserted foreign genetic material thatpreferably originates from a plant whose genome differs from that of theplant cells or tissues which contain the defective, non-infectious virusgenomes. Genetic material that is contemplated in this inventionincludes, but is not limited to, genes, which confer valuable propertiesto the plant, for example increased resistance such as increasedresistance to pathogens (e.g. phytopathogenic insects, fungi, bacteria,viruses etc.), to herbicides, insecticides or other biocides, toatmospheric influences or location conditions (e.g. heat, cold, wind,soil condition, moisture, dryness etc.), or with increased formation ofreserve or storage substances in leaves, seeds, tubers, roots, stalksetc. Hence regenerated plant cells or tissues can contain for examplegenomes of cauliflower mosaic virus with inserted foreign geneticmaterial, in particular material from a plant whose genome differs fromthat of the plant cells or tissues which contain cauliflower mosaicvirus.

A plant which contains cauliflower mosaic virus is, in the context ofthis invention, in particular Brassica rapa, for example Brassica rapacv Just Right.

The present invention also relates to regenerated plants which containdefective, non-infectious virus genomes and have been produced fromprotoplasts containing said virus genomes by culturing, including theirprogeny, which progeny may have been produced both by sexual or byvegetative propagation.

In another of its aspects, the invention relates to the use of agarosefor producing and culturing plant cells or tissues which containdefective, non-infectious virus genomes from isolated plant protoplastscontaining said virus genomes.

The invention relates further to the propagation of viral in vitroproduced mutants. Such mutants are propagated by infecting isolatedplant protoplasts with genetically manipulated viruses or virus genomes,and culturing the infected protoplasts in a suitable culture medium,preferably an agarose-solidified culture medium, followed by culturingin further suitable media until full differentiation to form a plant.Plant regeneration from cultural protoplasts is described in Evans, etal., "Protoplast Isolation and Culture," in Handbook of Plant CellCulture, 1:124-176 (MacMillan Publishing Co. New York 1983); M.R. Davey;"Recent Developments in the Culture and Regeneration of PlantProtoplasts," Protoplasts, 1983-Lecture Proceedings, pp. 19-29,(Birkhauser, Basel 1983); P. J. Dale, "Protoplast Culture and PlantRegeneration of Cereals and Other Recalcitrant Crops," in Protoplasts1983-Lecture Proceedings, pp. 31-41, (Birkhauser, Basel 1983).

A further object of the invention is the use in protoplasttransformation systems of viral in vitro produced mutants, which havebeen propagated as described above using genetically manipulated virusesor virus genomes.

Virus production in cell cultures formed from protoplasts can bedetected for example in two different ways: a) by hybridisation of thecell culture DNA with radioactively marked viral DNA or b) byreinfecting healthy plants with cell culture extracts.

Methods of isolating protoplasts and of detecting virus production areknown. However, it is advisable to vary the methods in accordance withthe respective conditions.

The production of isolated protoplasts will be described in more detailbelow, followed by Examples relating to protoplast and cellproliferation and to detecting virus production.

Brassica rapa plants (turnips) are reared in a greenhouse and, when inthe 5-leaf stage, infected with cauliflower mosaic virus by one of themethods commonly employed in virology (rubbing virus suspensions intoleaf surfaces by mechanical injury with carborundum powder).Systemically infected plants are transferred to a growth chamber forfurther culturing under the following conditions: 12/12 hours inlight/darkness rhythm, 5000 lux light intensity (SILVANIA daylightfluorescent tubes), 27° C. day temperature and 20° C. night temperature,70% constant relative humidity, twice daily treatment of the plantsgrowing in pots in an earth/sand/turf mixture with 0.1% of a plantfertiliser concentrate, e.g. Greenzit® fertiliser solution (CIBA-GEIGYAG, Basel). The protoplasts are routinely isolated from 12 cm longleaves of the systemically infected plants as follows: The leaves arewashed with tap water, sterilised for 30 minutes in 0.5% calciumhypochloride solution and then carefully washed 5 times in steriledeionised water under sterile conditions on a sterile work bench. Theleaf area is divided into 2 cm wide strips which are stacked and cutinto 1 mm wide cross-sections with ultra-sharp razor blades. The leafcross-sections are transferred to an osmotically stabilised enzymesolution of the following composition, with which they arevacuum-infiltrated: 1% cellulase ONOZUKA R10+0.1% pectinase MACEROZYMER10 (both supplied by Yakult Pharmaceutical Industry Co. Ltd., 8-21Shingikan-cho, Nishinomiya, Japan), dissolved in a mixture of 0.45 molarmannitol (3 vol) and 0.17 molar CaCl₂ (1 vol), pH adjusted to 5.4 with0.1 molar KOH. The osmotic pressure of the enzyme solution is about 510mOs/kg H₂ O. Most of the tissue is converted into isolated protoplastsby incubating the segments for 16 hours at 12° C. The protoplastsuspension is filtered through a 100 μm steel sieve to remove debris,transferred to sterile centrifuge tubes and sedimented for 10 minutes at75 g. The sedimented protoplasts are resuspended in sterile osmoticum(0.175 molar CaCl₂ +0.5% MES buffer (MES=2-(N-morpholino)ethanesulfonicacid; sigma No. M-8250), adjusted to pH 5.7, 510 mOs/kg H₂ O) and washed3 times by sedimentation at about 500× g and resuspension in theosmoticum. The protoplasts are then resuspended in the culture mediumdescribed in Example 1 and adjusted to a population density of 8×10⁴/ml.

The method used for protoplast transformation was as originallydeveloped by Krens et al. (1984), with modifications according toPaszkowski J, et al. (1984) 2·10⁶ protoplasts were used pertransformation treatment, each of which contained a total of 50 μg ofDNA (10 μg of the viral DNA and 30-40 μg of calf thymus DNA as carrier).

In particular this method involves contacting the DNA probe, comprisingthe probe molecule as well as a carrier DNA, with plant protoplast in asuitable osmotically stabilized incubation medium, such as, for example,but not limited to, K₃ -medium (Nagy JI and Maliga R, Z.Pflanzenphysiologie 78: 453-455, 1976; Shillito RD et al., MutationResearch 81: 165-175, 1981), containing polyethylene glycol with amolecular weight preferably between 1000 and 10000 g/Mol, morepreferably between 3000 and 8000 g/Mol, and in a concentration rangefrom 5% to 30% (w/v). The protoplasts and the DNA probe may be incubatedfor a period of 10 to 60 min, preferably for a period of about 30 min.

The incubation may be carried out in the light or, preferably, in thedark at a temperature between 18° C. and 32° C., preferably between 22°C. and 28° C.

EXAMPLE 1 Culturing protoplasts of Brassica rapa

Culture medium

modified according to Kao, K.N. (1977), Mol. Gen. Genet. 150, 225-230,and Koblitz, K. and Koblitz, D. (1982), Plant Cell Reports 1, 147-150.

    __________________________________________________________________________    NH.sub.4 NO.sub.3                                                                     600  mg/l                                                                              Na-pyruvate                                                                              5    mg/l                                         KNO.sub.3                                                                             1900 "   citrate    10   "                                            CaCl.sub.2.2H.sub.2 O                                                                 600  "   malate     10   "                                            MgSO.sub.4.7H.sub.2 O                                                                 300  "   fumarate   10   "                                            KH.sub.2 PO.sub.4                                                                     170  "   nicotinamide                                                                             0.5  "                                            KCl     300  "   pyridoxine-HCl                                                                           0.5  "                                            FeCl.sub.3.6H.sub.2 O                                                                 27   "   thiamine-HCl                                                                             5.0  "                                            Na.sub.2 EDTA                                                                         74.6 "   D-calcium pantho-                                                                        0.5  "                                                             tenate                                                       MnSO.sub.4.1H.sub.2 O                                                                 10   "   folate     0.2  "                                            Na.sub.2 MoO.sub.4.2H.sub.2 O                                                         0.25 "   p-aminobenzoicacid                                                                       0.01 "                                            H.sub.3 BO.sub.3                                                                      3.0  "   biotin     0.005                                                                              "                                            ZnSO.sub.4.7H.sub.2 O                                                                 2.0  "   choline chloride                                                                         0.5  "                                            CuSO.sub.4.5H.sub.2 O                                                                 0.025                                                                              "   riboflavin 0.1  "                                            CoCl.sub.2.6H.sub.2 O                                                                 0.025                                                                              "   ascorbic acid                                                                            1.0  "                                            KI      0.75 "   Vitamin D3 0.005                                                                              "                                            glucose 68'400                                                                             "                                                                sucrose 125  "                                                                fructose                                                                              125  "   Vitamin B12                                                                              0.01 "                                            ribose  125  "   m-inositol 100  "                                            xylose  125  "   casein hydro-                                                                            125  "                                                             lysate                                                       mannose 125  "   2,4-dichlorophen-                                                                        0.25 "                                                             oxyacetic acid                                               rhamnose                                                                              125  "   α-naphthylacetic-                                                                  0.5  "                                            Nitsch, J. P. and Nitsch. C (1969), Science 163, 85-87.                       KNO.sub.3                                                                             950  mg/l                                                                              m-inositol 100  mg/l                                         NH.sub.4 NO.sub.3                                                                     720  "   nicotinic acid                                                                           5    "                                            MgSO.sub.4.7H.sub.2 O                                                                 185  "   pyridoxine-HCl                                                                           0.5  "                                            CaCl.sub.2.2H.sub.2 O                                                                 220.5                                                                              "   thiamine-HCl                                                                             0.5  "                                            KH.sub.2 PO.sub.4                                                                     68   "   folic acid 0.5  "                                            FeCl.sub.3.6H.sub.2 O                                                                 27   "   biotin     0.05 "                                            Na.sub.2 EDTA                                                                         74.6 "   glycine    2                                                 MnSO.sub.4.1H.sub.2 O                                                                 17.25                                                                              "   agar (Difco)                                                                              9000                                                                              "                                            H.sub.3 BO.sub.3                                                                      10   "   sucrose    20000                                                                              "                                            ZnSO.sub.4.7H.sub.2 O                                                                 10   "   coconut milk (Gibco)                                                                     2.5  "                                            Na.sub.2 MoO.sub.4.2H.sub.2 O                                                         0.25 "   2,4-dichlorophen-                                                                        0.25 "                                                             oxyacetic acid                                               CuSO.sub.4.5H.sub.2 O                                                                 0.025                                                                              "   α-naphthylacetic                                                                   0.5  "                                                             acid                                                                          6-benzylamino-                                                                           0.1  "                                                             purine                                                       pH (KOH) 5.8; autoclaved for 20 min at 1 atom.                                __________________________________________________________________________

Culturing method

Cell colonies obtained in Example 1, which have attained a diameter of 1mm and more, are transferred to the surface of the above describedagar-solidified culture medium and further incubated at 24° C. inpermanent darkness.

In more than 20 experiments, protoplasts obtained from non-infectedBrassica rapa plants and from Brassica rapa plants infected withcauliflower mosaic virus were regenerated to cell cultures in the abovedescribed manner.

EXAMPLE 3 Detection of virus proliferation by hybridising the cellculture DNA with radioactively marked cauliflower mosaic virus DNA.

    ______________________________________                                        Aqueous solutions for the DNA hybridisation:                                  ______________________________________                                        proteinase K (Merck 24568):                                                                    0.1 mg/ml of proteinase K                                                     0.1% sodium azide                                                             0.1% sodium dodecyl sulfate                                                   (=SDS)                                                       alkali solution: 0.5M NaOH                                                                     1.5M NaCl                                                    neutral salt solution:                                                                         3M NaCl                                                                       0.5M tris(hydroxymethyl)amino-                                                methane buffer                                                                (=tris-HCl-buffer), pH 7.0                                   ______________________________________                                    

Method

a) individual cell colonies of Example 2 are each incubated in 1 ml of2-methoxyethanol for 5 hours at room temperature;

b) the 2-methoxyethanol is carefully removed by suction and the cellcolonies are frozen in liquid nitrogen;

c) the frozen cell colonies are homogenised to powder in Eppendorftubes;

d) the powdered material is suspended in 200 μl of H₂ O and thoroughlymixed;

e) the samples are centrifuged for 2 minutes in an Eppendorf centrifuge;

f) the clear supernatant is used for the DNA hybridisation.

Hybridisation

g) 50 μl portions of the supernatant [q.v. f) above] are put into thefilter chambers of the BRL dot hybridisation system (Bethesda ResearchLaboratories BRL No. 1050 MM), the filter of which must be moistenedbeforehand, and a mild vacuum is applied (water jet vacuum);

h) 200 μl of water are added and the samples are filtered;

i) 150 μl of the above proteinase K solution are put into each chamber,and the system is packed in plastic film and incubated overnight;

j) the proteinase solution is removed with suction, replaced by 150 μlof the above alkali solution and the samples are incubated for a further15 minutes;

k) the alkali solution is removed with suction and replaced by 150 μl ofthe above neutral solution and the samples are incubated for a further15 minutes;

l) after removal of the neutral salt solution, the filters are washedtwice for 5 minutes with 150 μl of sodium chloride/sodium citrate buffer(=SSC: 17.5 g of NaCl and 8.8 g of sodium citrate per liter of H₂ O; pHadjusted to 7 with NaOH);

m) after removal of the SSC, the filters are heated for 2 hours at 80°C.; and

n) subsequently hybridised with radio-active CaMV DNA;

o) radio-activity on the filters was visualised by exposure to a film;

p) extracts from cell cultures which have contained CaMV or CaMV DNAhybridise on the filter with the radio actively marked CaMV DNA and showup black on the processed film. From the presence or absence of blackareas on the film corresponding to the individual chambers of the BRLhybrid dot system it is possible to detect which of the tested cellcolonies contained CaMV or CaMV DNA.

EXAMPLE 4 Detection of virus proliferation by reinfection of healthyplants with cell culture extract

a) Individual cell colonies of Example 2 are each incubated in 1 ml of2-methoxyethanol for 5 hours at room temperature;

b) the 2-methoxyethanol is carefully removed with suction and the cellcolonies are frozen in liquid nitrogen;

c) the frozen cell colonies are homogenised to powder in Eppendorftubes;

d) each of the homogenised samples is suspended in 200 μl of H₂ O andthoroughly mixed;

e) the samples are centrifuged for 2 minutes in an Eppendorf centrifuge;

f) the clear supernatant is used for the reinfection assays.

Reinfection

The supernatant (q.v. f) above) is rubbed with carborundum, under strictsterile conditions, into the top side of leaves of healthy Brassica rapaplants. The plants are kept for 3 to 4 weeks free from the possibilityof any further virus infection and then examined for the occurrence ofvirus symptoms (mosaic symptoms, leaf vein transparency). Control plantstreated with carborundum are kept under the same conditions as theinoculated plants. The occurrence or absence of virus symptoms afterrubbing supernatant into the leaves of healthy plants is proof of thepresence or absence of viable CaMV particles in the cell culture fromwhich the supernatant has been obtained.

The results of the above described test showed that large amounts ofvirus have been formed in about 5% of the cell cultures which wereregenerated from protoplasts of virus-infected plants.

EXAMPLE 5 Use of non-infectious virus mutants as vectors for cell clonesin plant tissues.

Sap samples taken from different callus clones of Brassica rapacontaining CaMV DNA are rubbed into the top side of leaves of healthyBrassica rapa plants. The plants are kept for 3 to 4 weeks free from thepossibility of any other virus infection and then examined for theoccurrence of virus symptoms. 50% of the clones are infectious and theplants which have been treated with the sap of these clones exhibit theusual symptoms of a CaMV infection. The viral DNA of these plants has arestriction pattern which is identical with that of the viral DNA of thesap sample used for the infection.

30% of the clones are non-infectious and DNA analysis shows that thevirus genome exhibits deletions.

Based on the observation, that wild type CaMV can replicate in B.campestris var. rapa cell clones derived from infected plants and thatduring viral propagation in tissue culture its plant infectivity can belost (example 5), the following experiment (example 6), was carried outto prove that viral genomes, which are defective with respect toreplication in plants due to deletions or gene replacement, canreplicate in tissue culture.

EXAMPLE 6 Integration of genetically engineered CaMV genome into highmolecular weight plant genomic DNA

a) Construction of the plasmid pCaMV6Km

The plasmid pBR 327 CaKm⁺ described by Paszkowski et al., (1984), isdigested with restriction endonuclease EcoRV and the EcoRV restrictionfragment containing the kanamycin-resistant gene (NPT II) is used toreplace the EcoRV fragment of the plasmid pCa20-Bal I which fragmentcontains the gene VI of cauliflower mosaic virus, yielding the plasmidpCaMV6km. The plasmid pCa20Bal 1 is a chimaeric CaMV plasmid which isderived from the natural deletion mutant CM4-184 [Brisson, N. et al.(1984)]. The entire region II is missing from this plasmid, except forthe first 5 codons and the translation stop signal TGA. An XhoI couplingcomponent is inserted immediately before the stop codon in region II.All manipulations are carried out as described by Maniatis et al.(1982).

b) Protoplast transformation and culture

The method used for protoplast transformation was as originallydeveloped by Krens et al. (1982), with modifications according toPaszkowski J, et al. (1984). 1 ml aliquots of 2·10⁶ protoplasts are usedper transformation treatment, each of which are incubated for 30 min atroom temperature in K₃ medium (Nagy and Maliga, 1976; 0.1 mg/l 2.4-D, 1mg/l NAA, 0.2 mg/l BAP), containing 13% w/v PEG 6000 and a total of 50μg of DNA (10 μg of each plasmid molecule digested with Sal 1 in orderto release the viral DNA from the bacterial vector and 30-40 μg of calfthymus DNA as carrier). After transformation and washing of the DNA withF medium [Krens, F. A. et al., (1982); Krens, F. A. and Schilperoort, R.A., (1984)] with a pH of 5.3 (adjusted with KOH after autoclaving) theprotoplasts are collected by sedimentation (5 min at 100 g) andresuspended in 30 ml of K₃ -medium. Then the protoplasts are embedded in1.5% agarose (2.5·10⁵ protoplasts/ml) in a modified KO medium [Liang-caiL, Kohlenbach W, (1982)] containing 0.5 mg/l NAA, 0.25 mg/l 2,4-D, 0.1mg/l BAP, 520 mOs/kgH₂ O and cultured in an agarose bead type culturesystem [Shillito, R. D. et al., (1983)].

c) Selection of transformed cell clones

Selection is applied at day 4 of culture by the replacement of theculture medium surrounding the agarose beads with fresh mediumcontaining antibiotic [50 mg/l kanamycin, Jimenez, A. and Davis, J.(1980)].

After three weeks of culture with replacement of the selective medium at5 day intervals, the osmotic pressure of the culture medium is graduallyreduced to 200 mOs/kgH₂ O by reducing the mannitol concentration. After6 weeks visible resistant clones are picked up individually and arefurther cultured on 0.8% w/v agarsolidified KO-medium supplemented with50 mg/l kanamycin sulphate.

DNA isolation from plant material and Southern blot analysis

The methods and conditions for DNA isolation and hybridisation arecarried out as described previously [(EXAMPLE 3, Paszkowski J., et al.(1984)]

NPTII activity assay

NPTII activity is detected using the method described by Reiss et al.(1984) and adapted to plant material [Paszkowski J., et al. (1984)].

Infectivity and complementation test of engineered CaMV genome

CaMV DNA, amplified in E. coli by cloning on bacterial vector plasmids,is infectious on plants when cut out from the vector molecule andapplied to wounded leaves [Howell, S. H. et al., (1980); Lebeurier, G.et al. (1980)]. B. Campestris var. rapa is used in infectivity tests andin protoplast transformation experiments.

Infectivity is determined by inoculation of young plants with Sal 1restricted DNA of pCaMV6km or pCa20-Bal 1. DNA's are applied separatelyor as a 1:1 mixture. After 3 weeks, plants are examined for thedevelopment of CaMV specific mosaic symptoms. At the same time, DNA frominoculated leaves (site of infection) and from secondary infected leaves(infected by systemic spread) is examined by Southern blothybridisation, Southern, E. M. (1975) in order to follow the fate of theapplied DNA.

Application of pCaMV6km DNA alone never leads to the development ofmosaic symptoms. Infection with pCa20-Bal 1 DNA, or with mixtures ofboth DNA's cause the mosaic symptoms typical of systemic infection bythe wild-type virus.

Southern blot analysis, using specific probes which discriminate betweenthe two DNA's applied, reveals that pCaMV6km is not able to spreadsystemically through the plant. This is also true in the case wherepCaMV6km is applied together with pCa20-Bal 1 have never found pCa6kmspecific hybridisation signals in the secondary leaves of inoculatedplants.

Transformation of isolated turnip mesophyll protoplasts and selection ofantibiotic resistant cell lines

In order to study the possibility of pCaMV6km replication at thecellular level in cultured plant cells turnip protoplast transformationexperiments are undertaken.

Recently turnip mesophyll protoplast culture has been improved to c.a.20% plating efficiency [Pisan, B. et al. (1983)], a level sufficient toapproach DNA transformation. For protoplast transformation amodification of a DNA uptake procedure previously developed by Krens etal. (1982) is used. Readjustment of the pH of F medium [Krens et al.,(1982)] to 5.5, which falls to 4.8 after autoclaving, is a prerequisitefor survival of turnip protoplasts during the DNA treatment. Even so,recovery of protoplasts after transformation is not satisfactory in themajority of experiments. In only 4 out of 15 equivalent experiments wasthe plating efficiency at a level (10-15%) sufficient to carry outselection of transformants. Antibiotic resistant cell lines arerecovered in 2 of these experiments.

We obtained 2 clones from kanamycin sulphate selection. These clones aregrown further on agar-solidified media containing 50 mg/l kanamycinsulphate. As soon as the clones reached a size of approximately 1 gfresh weight of tissue, part of the material is subjected to Southernblot analysis and assayed for the presence of the NPTII gene product.

Form and arrangement of the foreign DNA in transformed kanamycinresistant cell lines

DNA of the two clones selected and cultured on kanamycin-containingmedia (T12A and T12B) are analysed after 3, 6, 9 and 12 months ofculture-the foreign DNA was stably maintained. When non restricted totalcellular DNA is analysed, hybridisation with radioactively labelledpCaMV6km is detected only in the region of high molecular weight DNAgreater than 50 kilobases in length. There is no detectablehybridisation in the region of 7.1 or 7.5 kb, the expected sizes forfree copies of viral like molecules derived from plasmids pCaMV6km andpCa20-Bal 1, respectively. Further analysis of nuclear DNA confirmed thesupposition that the major part of the Sal 1 fragment of pCaMV6kmcontaining the hybrid viral genome had been integrated, generatingjunction fragments with plant DNA in both of the selected cell linesT12A and T12B. Reconstructions and mapping experiments suggested thepresence of a single copy of the transforming DNA molecule per diploidgenome of B. campestris var. rapa. The results of the Southern blotanalyses suggest the following conclusions on the junctions of pCaMV6kmDNA and plant DNA:

a) The 114 bp EcoRV fragment containing the hybrid marker gene ispresent in a non-rearranged form, which when correlated to tests forNPTII activity, can be taken as evidence for the integration of afunctional copy of this gene into the plant genome.

b) EcoRV, BstEII and EcoRI restriction digests of nuclear DNA of T12Aand T12B hybridised to probes specific for either the NPTII codingregion or the 5' and 3' gene flanking sequences allowed for approximatemapping of the regions of the integrated pCaMV6km Sal 1 fragment. Thefragments of foreign DNA integrated in two transformed lines aredifferent and are probably integrated at different sites in the genome(as judged from the sizes of the border fragments). There are, however,also common features, in that both lines contain an unaltered (2297 bplong) EcoRI fragment of the linear molecule used for transformation andthere is little rearrangement of the transforming DNA.

NPTII activity test of transformed cell lines

Kanamycin-specific phosphorylation, reflecting activity of NPTII, can bedetected in crude plant protein extracts electrophoresed under nondenaturing conditions in a 10% polyacrylamide gel.

Extracts of kanamycin resistant cell lines are tested for the presenceof the NPTII activity.

Extracts of the kanamycin resistant cell lines (T12A and T12B) containthe expected NPTII specific activity, confirming the biological functionof the integrated hybrid marker gene. Moreover the banding pattern ofthe enzyme is identical to that found in tobacco tissues expressing theenzyme from the same fused gene.

Analogous experiments have been carried out with plants of the speciesBrassica napus (rape).

The regeneration of Brassica napus protoplasts is achieved by use of amethod described by Liang-cai Li and Kohlenbach, (1982).

EXAMPLE 7 Cross-protection in CaMV producing transgenic plants

Plants which are systemically infected with a virus are resistant to asecondary infection by a related virus strain. This phenomenon is calledcross-protection but the exact mechanism is still unknown (Sherwood,CIBA Foundation Sumposium, 133:136-150 (1987)). In order to determinewhether 1050- and 1123-transgenic plants exhibit cross-protection, theseplants are superinfected by another CaMV strain. Transformed plants areinoculated with a CaMV strain, namely Ca-NB2, that has gene II replacedby a DHFR gene (dehydrofolate reductase gene) and thereby allowingdistinction from virus produced by the transformed plants. Ca-NB2 hasbeen constructed and tested for normal infectivity by Brisson et al.,Nature, 310:511-514 (1984). Four 1123, four 1050 and two 1051transformed (nopaline positive) plants are inoculated with extract ofturnip leaves, systemically infected with Ca-NB2. Four weeks after thesuperinfection total DNA is extracted from the infected plants and fromuninfected plants. The DNA is slot blotted to a Zeta probe membrane,hybridized with a ³² P-labelled DHFR-specific probe and the membraneexposed to an x-ray film. The slot blot analysis shows that there is noreplication of Ca-NB2 in 1050 transgenic plants and no or very littlereplication in 1123 transgenic plants which indicates that the plantsare protected against secondary infection by Ca-NB2. The membrane iswashed free of the DHFR-specific probe and then incubated with aCaMV-DNA-specific probe. The resulting autoradiogram shows that there isvirus present in the superinfected transgenic plants. Progeny plantstransgenic for the "recombination CaMV-construct" (1123) produce noinfectious virus in cotyledons. Only in leaves that appear later,usually in the second leaf or sometimes much later, is infectious CaMVfound, shown by slot blot analysis of total plant DNA or by infectivitytests on turnip plants. Progeny plants of 1050, however, produceinfectious CaMV already in the cotyledons. This observation may be areason for the slightly less complete cross-protection in 1123 plantscompared to the 1050 plants. Another reason could have been thedifferent CaMV strains used. The CaMV-DNA in construct 1050 consistsmainly of strain 4-184 (gene VI of strain S) like the challenging virus(Ca-NB2), whereas construct 1123 consists of CaMV strain DH sequences.

The "cross-protection" observed in the above experiment can beattributed to recombination between Ca-NB2 and the virus produced by thetransgenic plants to CaMV lacking the DHFR gene. Alternatively, the CaMVstrains produced by the transgenic plants may replicate at a greaterrate and thus out-compete the incoming Ca-NB2 virus. In order to testthe ability of Ca-NB2 to compete in systemic spreading with strain DH(in construct 1123) or 4-184 (in construct 1050), leaf extractcontaining Ca-NB2 is inoculated with extract containing either of theother strains at the same time on turnip plants. The different virusextracts are either rubbed together on the same leaf or on separateleaves; once Ca-NB2 extract is inoculated on the younger leaf of aturnip and the 1050 extract on the following older leaf or vice versa.Isolation of total plant DNA and analysis by the slot blot techniqueusing a DHFR-specific probe for DNA hybridization, shows that Ca-NB2virus is present in high amounts. No differences are observed betweenthe ways the double infections are performed.

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What is claimed is:
 1. A process for the maintenance and proliferationof defective, non-infectious CaMV genomes in proliferating plantmaterial, said CaMV genomes being unaccompanied by the T-DNA borderregions of the Ti-plasmid, said process comprising:culturing in asuitable nutrient medium proliferating isolated plant protoplasts,isolated plant cells or isolated plant tissues containing non-infectiousCaMV genomes, said CaMV genomes being unaccompanied by the T-DNA borderregions of the Ti-plasmid and being stably integrated into the plantgenome, which CaMV genomes are not viable under natural conditions andhave lost their ability to replicate in plant material.
 2. A processaccording to claim 1, wherein the defective, non-infectious CaMV genomesare viral mutants selected from the group consisting of deletionmutants, substitution mutants, insertion mutants and mutants havingrearranged viral genes.
 3. A process according to claim 1, wherein thedefective, non-infectious CaMV genomes are genetically modifiedcontaining non-viral genetic material heterologous with respect to theplant genome.
 4. A process according to claim 1, which processcomprises:(a) isolating plant protoplasts; (b) introducing a defectivenon-infectious CaMV genome into the genome of the plant protoplasts byincubation with polyethylene glycol; and (c) cultivating saidprotoplasts in a suitable nutrient medium.
 5. A process according toclaim 1, which process comprises:(a) isolating plant protoplasts bytreating appropriate plant material derived from leaves, stems,blossoms, roots, pollen, cotyledons, seedlings or seeds with suitablecellulase and pectinase enzymes; (b) introducing a defective,non-infectious CaMV genome without the T-DNA border regions of theTi-plasmid into said isolated plant protoplasts, by incubation withpolyethylene glycol, while maintaining said plant protoplasts and theCaMV genome in a suitable incubation medium; (c) cultivating said plantprotoplasts containing the defective, non-infectious CaMV genome in asuitable nutrient medium in a temperature range from 7° C. to 42° C. andat a protoplast density of from 1 to 1×10⁶ protoplasts/ml. 6.Proliferating isolated plant protoplasts, plant cells or plant tissues,which contain stably integrated into their genomes defective,non-infectious CaMV genomes.
 7. Proliferating isolated plantprotoplasts, plant cells or plant tissues according to claim 6, whereinthe defective, non-infectious CaMV genomes are genetically modified CaMVgenomes.
 8. Proliferating isolated plant cells or plant tissues, whichhave been regenerated from plant protoplasts and plant cells,respectively, according to claim 6, which plant protoplasts or plantcells are derived from plants that are capable of being regenerated. 9.Proliferating isolated plant cells or plant tissues, which have beenregenerated from plant protoplasts or plant cells, respectively,according to claim 7, which protoplasts or cells are derived from plantsthat are capable of being regenerated.
 10. A process according to claim1, which process comprises:(a) isolating plant protoplasts by treatingappropriate plant material derived from leaves, stems, blossoms, roots,pollen, cotyledons, seedlings or seeds with suitable cellulase andpectinase enzymes; (b) introducing a defective, non-infectious CaMVgenome, said CaMV genome being unaccompanied by the T-DNA border regionsof the Ti-plasmid, into said isolated plant protoplasts by incubationwith polyethylene glycol, while maintaining said plant protoplasts andthe CaMV genome in a suitable incubation medium; (c) transferring saidplant protoplasts containing the defective, non-infectious CaMV genomeinto a suitable liquid nutrient medium, solidifying said medium withagarose, cutting the agarose-solidified medium into segments aftersetting, and transferring the segments to a suitable nutrient medium;and (d) cultivating said segments in a temperature range from 7° C. to42° C.